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Adjuvant-Enhanced Antibody Responses in the Absence of Toll-Like Receptor Signaling

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Science  22 Dec 2006:
Vol. 314, Issue 5807, pp. 1936-1938
DOI: 10.1126/science.1135299

Abstract

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell–dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.

Adjuvants are vaccine additives that enhance the elicited levels of antibodies and T lymphocyte responses by promoting inflammatory responses of leukocytes in ways that presumably mimic natural infection. Toll-like receptor (TLR)–mediated recognition of microbial signature molecules is one of the cues normally used by leukocytes to react to real microbial challenges (1, 2). Each of the 10 different functional TLRs in humans (12 in mice) has apparently evolved to recognize a specific set of evolutionarily conserved molecules, including components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA (1, 2). TLR recognition leads to the activation of transcription factors that drive cytokine expression, proliferation, survival, and inflammatory mediator expression. TLR signaling is initiated by four adapters—MyD88, Toll-interleukin 1 receptor (TIR) domain–containing adapter inducing interferon beta (TRIF), TIR domain–containing adapter protein (TIRAP)/MyD88 adapter–like (Mal), and TRIF-related adapter molecule (TRAM)—which associate with the cytoplasmic TIR domains of TLRs (1, 2). MyD88 associates with all TLRs except TLR3, whereas TRIF associates with TLR3 and TLR4. TIRAP/Mal and TRAM appear to function as bridging adapters for MyD88 and TRIF, respectively (1, 2). TIRAP/Mal and TRAM are essential for signaling by TLR4, with TIRAP/Mal also required for TLR2 function. MyD88 also contributes to signaling in B cells and is required for maximal B cell responses to foreign proteins when present in the context of TLR ligands (3). Nevertheless, there is debate about whether such signals are necessary for this class of response (4, 5). Mice genetically deficient in both MyD88 and TRIF (Myd88–/–;Trif Lps2/Lps2 mice) have a complete lack of known TLR signaling (68), thus allowing an assessment of the TLR dependence of antibody responses. We took advantage of this to explore more precisely the role of TLR signaling in antibody responses to immunization and the augmenting roles of adjuvants in this response.

Myd88–/–;Trif Lps2/Lps2 and control C57BL/6 mice were immunized with the T cell–dependent antigen trinitrophenol-hemocyanin (TNP-Hy) given in Freund's complete adjuvant (FCA), and the titers of induced antibodies to TNP in the serum were determined (9). In these analyses, antibody responses of Myd88–/–;Trif Lps2/Lps2 mice were entirely comparable to those of C57BL/6 mice, indicating that signals transmitted by TRIF and MyD88 made no appreciable contribution to the antibody response (Fig. 1). This experiment included initial immunization, followed by a boost with TNP-Hy in phosphate-buffered saline (PBS) on day 21, and showed no significant defect in sera at any time point for immunoglobulin M (IgM), IgG1, IgG2b, IgG2c, IgG3, and IgE antibody responses to TNP. Furthermore, when the TNP-Hy challenge was given with the adjuvant alum, a frequently used adjuvant in human vaccines, antibody responses of Myd88–/–;Trif Lps2/Lps2 mice were also comparable to those of C57BL/6 mice (5) (fig. S1).

Fig. 1.

T cell–dependent antibody responses of Myd88–/–;TrifLps2/Lps2 and control mice using FCA. Two-month-old mice were immunized with TNP-Hy in FCA on day 0 and boosted with antigen in PBS on day 21. Each point represents the serum antibody titer to TNP for an individual mouse. IgE titers represent ng/ml, whereas other measurements represent reciprocal serum dilution yielding half-maximal signal. Closed circles, wild-type C57BL/6; open squares, Myd88–/–;TrifLps2/Lps2 double knockout mice. *, P < .05; **, P < .01. For methods, see (9).

To reevaluate the augmenting effects of adjuvant on antibody production and its suggested dependence on MyD88 and TRIF, additional immunizations of C57BL/6 and of Myd88–/–;Trif Lps2/Lps2 mice were carried out with a second antigen, TNP–keyhole limpet hemocyanin (TNP-KLH), in which the adjuvants FCA, Freund's incomplete adjuvant (FIA), and monophosphoryl-lipid A/trehalose dicorynomycolate (“Ribi” adjuvant) were compared with responses in the absence of adjuvant. TNP-specific antibody responses were assessed 7 and 14 days later (Fig. 2). Ribi adjuvant contains the TLR4 ligand monophosphoryl-lipid A, whereas FIA is not known to contain any TLR ligand. Both C57BL/6 and Myd88–/–;Trif Lps2/Lps2 mice responded strongly to TNP-KLH only when given in adjuvant (Fig. 2). [The IgG1 responses to TNP-KLH given in PBS, although low, were significantly above preimmune background (titer < 10)]. Responses of Myd88–/–;Trif Lps2/Lps2 mice to antigen given in FCA or FIA were unimpaired, whereas responses to TNP-KLH given in Ribi adjuvant were reduced relative to those of wild-type mice at day 14 (Fig. 2). However, the reduction in antibody titers elicited in Myd88–/–;Trif Lps2/Lps2 mice was modest and observed mainly in the IgG2c and IgG2b responses (Fig. 2). We conclude that, under these experimental conditions, TLR signaling is not required for T cell–dependent antibody responses and makes a relatively small contribution to responses to antigen given in either alum or FCA.

Fig. 2.

Comparison of serum antibody responses of C57BL/6 and Myd88–/–;TrifLps2/Lps2 mice given immunogen in different adjuvants. Groups of four mice were immunized with 100 μg TNP-KLH in PBS in the indicated adjuvants, and titers of antibody to TNP of the indicated immunoglobulin isotypes were determined on days 7 and 14.

It was notable that even in mice challenged with Ribi adjuvant, which contains a potent TLR ligand, most of the antibody-augmenting adjuvant effect was independent of MyD88/TRIF (Fig. 2). Consistent with this, Ribi, but not FIA or FCA, stimulated peritoneal macrophages to produce type I interferon (IFN) and tumor necrosis factor (TNF) when used to treat cells at concentrations approximating those achieved in vivo (fig. S2). As expected, isolated splenic B cells from Myd88–/–;Trif Lps2/Lps2 mice failed to proliferate in response to the TLR ligand lipopolysaccharide (LPS) (fig. S3B). By contrast, responses of Myd88–/–;Trif Lps2/Lps2 B cells to B cell receptor ligation were indistinguishable from those of normal cells (fig. S3, A and C). Nevertheless, it was apparent that the observed adjuvant effect on antibody responses in vivo failed to correlate with their potential to activate myeloid cells or B cells in vitro.

Thus far, the responses we had measured were for antigens that depend on T cell help, although another class of antibody response is independent of help from T cells. Myd88–/–;Trif Lps2/Lps2 mice were thus tested for their responses to the T cell–independent type 2 (TI-2) antigen TNP-Ficoll. Overall antibody responses did not generally differ significantly from normal controls (fig. S4). These results show for the first time that the antibody response to a typical TI-2 antigen is independent of TLR signaling.

Preimmune serum levels of Ig in Myd88–/–;Trif Lps2/Lps2 mice were next analyzed and found to have a complex pattern, with some isotypes reduced in amount and others increased relative to C57BL/6 levels (Fig. 3). Relative to control, IgG2b and IgG2c levels were lower in Myd88–/–;Trif Lps2/Lps2 mice, by 50% and 70%, respectively, a statistically significant but modest reduction (Fig. 3). Also, the serum levels of one Ig subclass (IgG3) in Myd88–/–;Trif Lps2/Lps2 mice were notably lower. By contrast, Myd88–/–;Trif Lps2/Lps2 mice displayed 3 times the levels of IgG1 (P < .001) and 20 times the levels of IgE (P < .0001). Collectively, such differences suggest that TLR signaling may control the class rather than the magnitude of Ig levels in naive mice, at least under specific pathogen-free conditions such as those found in our facility.

Fig. 3.

Preimmune serum immunoglobulin levels in Myd88–/–;TrifLps2/Lps2 compared with wild-type C57BL/6 controls as measured by enzyme-linked immunosorbent assay. Each point represents data from a different mouse. Closed circles, wild-type, C57BL/6; open squares, Myd88–/–;TrifLps2/Lps2 mice.

Consistent with the robust preimmune Ig levels and antibody responses of Myd88–/–;Trif Lps2/Lps2 mice, nearly normal B-1, marginal zone, and follicular B cell numbers were found in these animals. Compared with wild-type mice, the only consistent differences found in Myd88–/–;Trif Lps2/Lps2 mice tested at 2 and 6 months of age were a B cell surface level of CD23 about twice as high (fig. S5) and a slight reduction in splenic transitional type 1 B cells (T1) (tables S1 and S2). We conclude that TLR signaling is not required for preimmune B cell development but may partly affect the abundance and CD23 levels of B cell subsets. As levels of CD23, IgE, and IgG1 are up-regulated by interleukin 4, we speculate that environmental stimuli transduced by TLRs in a MyD88- and/or TRIF-dependent manner suppress the expression of, or response to, this cytokine.

Our findings reveal that TLR signaling is not required for robust antibody responses to antigen when given in four commonly used adjuvants, in particular FCA, which is widely thought to depend on TLR signaling for its adjuvant effect (10). However, ligands that signal through the MyD88 and TRIF pathways can costimulate these responses and affect the antibody class of the response, as has been known for LPS for many years (3), and is indicated by the modest boost seen in C57BL/6 mice immunized with Ribi adjuvant (Fig. 2). By exclusion, our data suggest the likelihood that non-TLR–mediated “innate” signals may be involved in the augmentation of adaptive antibody responses. It is also formally possible that MyD88/TRIF–independent modes of TLR signaling are yet to be discovered, possibly through TRAM and TIRAP, although so far there is no evidence for this.

Our results extend and clarify previous studies. A recent paper involving MyD88-deficient mice asserted that T cell–dependent antibody responses require activation of TLRs in B cells (4). However, that inference was based on the use of LPS as an adjuvant rather than more commonly used adjuvants such as FCA or alum. Indeed, an earlier study found that although MyD88-deficient mice failed to make an IgG2a response to ovalbumin given in FCA, they still made good IgG1 and IgE antibody responses, and antigen given in alum could promote an IgE response (11). However, in those studies, the remaining antibody responses in MyD88-deficient mice could conceivably have involved TLR signaling through TRIF-dependent pathways, a caveat that does not apply to our data. We clearly find that IgG2c (also known as IgG2ab) responses to protein antigen given in alum or FCA are robust in Myd88–/–;Trif Lps2/Lps2 mice, in apparent contradiction to both studies of MyD88-deficient mice mentioned (4, 11) and other studies implicating a requirement of MyD88 or TLR signaling in the generation of IgG2a antibodies. It may be that TLR ligand–driven suppression of IgG2c/a production occurs in mice with intact TRIF signaling that lack MyD88; for example, through cytokine-driven polarization of the T helper response (11, 12). The IgG2c/a response is promoted by IFN-α/β or IFN-γ (13, 14). In viral infection, the IgG2c/a response is lost in mice lacking both IFN-α/βR and IFN-γR (15). Because we found good IgG2c responses upon immunization of Myd88–/–;Trif Lps2/Lps2 mice, our results may indicate that these immunizations can stimulate TLR-independent IFN production.

The antibody response to the T cell–independent type II antigen TNP-Ficoll was relatively normal in Myd88–/–;Trif Lps2/Lps2 mice, although it was slightly lower at later time points. Interestingly, the IgG3 component of the antigen-specific response of Myd88–/–;Trif Lps2/Lps2 mice was not significantly lower than normal despite their reduced preimmune total serum IgG3 levels. It has been argued that the class switch to IgG3 requires cytokines produced by accessory cells (16). If this is so, it is clear from these results that production of the cytokines in question does not require TLR stimulation of B cells or accessory cells.

That B cells can respond to autologous DNA and RNA through TLR signaling (17, 18) raised the possibility that TLRs could affect preimmune development or maintenance of B cell subsets, particularly the marginal zone and B-1 compartments. However, our findings that Myd88–/–;Trif Lps2/Lps2 mice generate abundant B-1 and marginal zone B cells appear to rule out definitively a required role for TLR signaling in their development.

Our data do not contest the long-held understanding that TLR ligands can augment antibody responses. However, it is surprising, given the recent emphasis on the importance of TLRs in the initiation of the adaptive immune response, that we fail to find a deficit in the early antibody responses of Myd88–/–;Trif Lps2/Lps2 mice using conventional antigens and immunization regimens. Our data are more consistent with a model in which TLRs play roles in early microbial suppression, regulation of the antibody class, and sustaining antibody secretion at late times after immunization, rather than as an essential component of the self/nonself discrimination of theadaptiveimmuneresponse. Thesedatahave implications for vaccine design because they indicate that robust antibody responses to moderate doses of antigens can be achieved when given in the total absence of TLR ligands. Because TLR-mediated signals can be toxic, our findings raise the possibility that unwanted side effects of adjuvants may be avoided by excluding TLR ligands from adjuvants.

Supporting Online Material

www.sciencemag.org/cgi/content/full/314/5807/1936/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 and S2

References

References and Notes

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